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Analytical and Bioanalytical Chemistry (v.390, #4)

Aptamers and their applications by Marco Mascini (pp. 987-988).

has been one of the pioneers of biosensor technology in Europe. More than 400 of his papers on this subject have been published in the past 35 years and he is presently engaged in the exploitation of short and large sequences of nucleotides as elements for the molecular recognition for biosensors. The recent applications include detection of medical diagnostics, environmental compounds, genotoxic effects, biological terrorist threat agents and food quality and safety.

Aptamers: molecular tools for analytical applications by Teresa Mairal; Veli Cengiz Özalp; Pablo Lozano Sánchez; Mònica Mir; Ioanis Katakis; Ciara K. O’Sullivan (pp. 989-1007).

Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.

Keywords: Aptamers; Aptasensors; SELEX; Affinity chromatography; Spiegelmers; Aptamer molecular beacons

Surface immobilization methods for aptamer diagnostic applications by Subramanian Balamurugan; Anne Obubuafo; Steven A. Soper; David A. Spivak (pp. 1009-1021).

In this review we examine various methods for the immobilization of aptamers onto different substrates that can be utilized in a diverse array of analytical formats. In most cases, covalent linking to surfaces is preferred over physisorption, which is reflected in the bulk of the reports covered within this review. Conjugation of aptamers with appropriate linkers directly to gold films or particles is discussed first, followed by methods for conjugating aptamers to functionally modified surfaces. In many aptamer-based applications, silicates and silicon oxide surfaces provide an advantage over metallic substrates, and generally require surface modification prior to covalent attachment of the aptamers. Chemical protocols for covalent attachment of aptamers to functionalized surfaces are summarized in the review, showing common pathways employed for aptamer immobilization on different surfaces. Biocoatings, such as avidin or one of its derivatives, have been shown to be highly successful for immobilizing biotin-tethered aptamers on various surfaces (e.g., gold, silicates, polymers). There are also a few examples reported of aptamer immobilization on other novel substrates, such as quantum dots, carbon nanotubes, and carbohydrates. This review covers the literature on aptamer immobilization up to March 2007, including comparison of different linkers of varying size and chemical structure, 3′ versus 5′ attachment, and regeneration methods of aptamers on surfaces.

Keywords: Aptamer; Sensor; Immobilization; Linker; Optimization; Assay

Aptamers as molecular recognition elements for electrical nanobiosensors by Jeong-O Lee; Hye-Mi So; Eun-Kyoung Jeon; Hyunju Chang; Keehoon Won; Yong Hwan Kim (pp. 1023-1032).

Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors. This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements. The advantages of aptamers over antibodies as sensors are highlighted. These advantages are especially apparent with electrical sensors such as electrochemical sensors or those using field-effect transistors. Figure Feeling proteins with aptamer-functionalized carbon nanotubes

Keywords: Aptamers; Nanobiosensors; Electrochemical aptamer sensors; Field-effect transistor aptamer sensors

Monitoring the progression of the in vitro selection of nucleic acid aptamers by denaturing high-performance liquid chromatography by Jens Müller; Osman El-Maarri; Johannes Oldenburg; Bernd Pötzsch; Günter Mayer (pp. 1033-1037).

Monitoring of in-vitro selection experiments is crucial in evaluation of the success and outcome of such approaches. Furthermore, monitoring running in parallel with the selection procedure enables early intervention and adjustment of stringency to achieve the desired activities of the selected nucleic acid species. Here we describe the use of a non-radioactive method that enables monitoring of a SELEX procedure on the basis of sequence diversity. We employ denaturing HPLC and describe for the first time an experimental set-up that is useful both for analysis of the progression of in-vitro selection experiments and for separation of distinct aptamer sequences.

Keywords: Aptamer; SELEX; dHPLC WAVE

DNA aptamers against the MUC1 tumour marker: design of aptamer–antibody sandwich ELISA for the early diagnosis of epithelial tumours by C. S. M. Ferreira; K. Papamichael; G. Guilbault; T. Schwarzacher; J. Gariepy; S. Missailidis (pp. 1039-1050).

Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer–antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC₅₀ value of 25 μg/ml, a detection limit of 1 μg/ml and a linear range between 8 and 100 μg/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours. Figure An aptamer-antibody two-dimentional immunoassay for MUC1

Keywords: MUC1; Aptamers; ELISA; Diagnostic assay; Immunoassay

Covalently bonded DNA aptamer chiral stationary phase for the chromatographic resolution of adenosine by Joséphine Ruta; Corinne Ravelet; Jérôme Désiré; Jean-Luc Décout; Eric Peyrin (pp. 1051-1057).

In this work, a target-specific aptamer chiral stationary phase (CSP) based on the oligonucleotidic selector binding to silica particles through a covalent linkage was developed. An anti-d-adenosine aptamer was coupled, using an in-situ method, by way of an amide bond to macroporous carboxylic acid based silica. Frontal chromatography analysis was performed to evaluate the column properties, i.e., determination of the stationary phase binding capacity and the dissociation constant of the target-immobilized aptamer complex. It was found that such covalent immobilization was able to maintain the aptamer binding properties at a convenient level for an efficient enantioseparation. Subsequently, the separation of adenosine enantiomers was investigated under different operating conditions, including changes in the eluent’s ionic strength and the proportion of organic modifiers as well as column temperatures. It was demonstrated that, under various conditions of use and storage, the present CSP was stable over time.

Keywords: Chiral stationary phase; Aptamers; Adenosine enantiomers

Characterizing the interaction between aptamers and human IgE by use of surface plasmon resonance by Jinli Wang; Renji Lv; Jingjuan Xu; Danke Xu; Hongyuan Chen (pp. 1059-1065).

Human immunoglobulin E (hIgE) is such an important protein, because of its involvement in allergic disease, that it is of significance to study the interactions between it and its recognizing elements. In this report an analytical strategy based on surface plasmon resonance (SPR) was developed to probe the pattern of interaction between hIgE and its recognizing molecules, including aptamers and antibodies. The affinity constants of hIgE for the antibody and the aptamer were compared first; the aptamer has more affinity than the antibody for human IgE. To study their pattern of interaction, three different binding approaches, including adding the antibody and the streptavidin-coupled aptamer to the sensing surface, were designed. The results showed that hIgE captured on the sensing surface could form a multivalent complex with the aptamer. An ELISA-like assay using the aptamer as both capture and detection probes was then developed. This work highlights an SPR method for characterizing the interaction between the protein and aptamers that is useful for study of biomolecular interaction patterns and binding properties. Figure Schematic diagram of the use of surface plasmon resonance for detection of the pattern of interaction of human IgE with its DNA aptamer and antibody

Keywords: Aptamer; Surface plasmon resonance; Affinity constants; Interaction pattern

Selection of fluorescent aptamer beacons that light up in the presence of zinc by Manjula Rajendran; Andrew D. Ellington (pp. 1067-1075).

In order to generate nucleic acid biosensors that could undergo a reversible conformation change in the presence of the metal zinc, a random sequence pool of single-stranded DNA was immobilized on an oligonucleotide affinity column. In the presence of zinc, those species that underwent a conformational change were released from the column, collected, and amplified. A series of negative and positive selections refined the metal specificity of the selected aptamer beacons. Since the aptamer beacons contained a fluorophore, while the bound oligonucleotide contained a quencher, zinc binding also resulted in an increase in fluorescence. One of the selected beacons, Zn-6m2, bound zinc in the low micromolar range, gave a dose-dependent fluorescence signal, and showed an approximately sixfold increase in fluorescence on zinc binding. While some cross-reactivity with cadmium was observed, it should nonetheless prove possible to use the novel selection method to generate and tune the specificity of a variety of reversible metal biosensors. Such biosensors could potentially be used for continuous monitoring of metals in environmental samples.

Keywords: Aptamer; In vitro selection; SELEX; Biosensor; Zinc; Conformational switch

Development of an optical RNA-based aptasensor for C-reactive protein by A. Bini; S. Centi; S. Tombelli; M. Minunni; M. Mascini (pp. 1077-1086).

The development of a RNA-aptamer-based optical biosensor (aptasensor) for C-reactive protein (CRP) is reported. CRP is an important clinical biomarker; it was the first acute-phase protein to be discovered (1930) and is a sensitive systemic marker of inflammation and tissue damage. It has also a prognostic value for patients with acute coronary syndrome. The average concentration of CRP in serum is 0.8 ppm and it increases in response to a variety of inflammatory stimuli, such as trauma, tissue necrosis, infection and myocardial infarction. The interaction between the 44-base RNA aptamer and the target analyte CRP is studied. In particular, the influence of the aptamer immobilization procedure (chemistry, length, concentration), as well as the binding conditions, i.e., the influence on the binding of different buffers, the presence of Ca²⁺ ion and the specificity (against human serum albumin) have been evaluated. Using the best working conditions, we achieved a detection limit of 0.005 ppm, with good selectivity towards human serum albumin. Some preliminary experiments in serum are reported. Figure The assay on the CM5 chip

Keywords: Aptamer; C-reactive protein; Surface plasmon resonance

The study of surface properties of an IgE-sensitive aptasensor using an acoustic method by M. Šnejdárková; L. Svobodová; V. Polohová; T. Hianik (pp. 1087-1091).

We applied the acoustic transverse shear mode (TSM) method for study of the surface properties of a DNA aptasensor that specifically binds human immunoglobulin E (IgE). The biotinylated 45-mer DNA aptamers were immobilized on the surface of a self-assembled layer composed of a mixture of polyamidoamine dendrimers of the fourth generation with 1-hexadecanetiol covered by neutravidin. Using the TSM method, we studied the kinetics of changes of the series resonant frequency, f _s, and the motional resistance, R _m, of a quartz crystal transducer, used as a support for formation of the sensing layer. We have shown that attachment of the biotinylated DNA aptamers onto the surface covered by neutravidin results in a decrease of f _s, but in an increase of R _m. Similar changes of f _s and R _m were observed following addition of IgE. This suggests the contribution of friction forces to the crystal oscillation, which was taken into account in the calculation of the mass changes at the sensor surface following binding processes.

Keywords: DNA aptamer; Immunoglobulin E; Dendrimers; Transverse shear mode method; Biosensor

Study of thiazole orange in aptamer-based dye-displacement assays by Renjun Pei; Milan N. Stojanovic (pp. 1093-1099).

We screened a series of RNA and DNA aptamers for their ability to serve in the dye displacement assays in which analytes compete with TO dye. We conclude that, while the performance of the TO dye displacement approach is not always predictable, it is still a simple and sensitive assay to detect binding between RNA aptamers and small molecules. In particular, we describe efficient assays for tobramycin and theophylline, with up to 90% displacement of TO observed, and we describe the first aptameric assay for cAMP. Figure An RNA or DNA aptamer against a molecule (circle) binds TO dye, resulting in a fluorescent complex. Presence of free molecule in solution results in the displacement of TO from the complex and a reduction in fluorescence

Keywords: Aptamers; Fluorescence assays; Thiazole orange; Displacement assays

Chromatographic characterization of molecularly imprinted polymers by Wen-Chien Lee; Chung-Hsien Cheng; Hsin-Hung Pan; Ting-Hao Chung; Ching-Chiang Hwang (pp. 1101-1109).

Recent efforts in the investigation of chromatographic characterization of molecularly imprinted polymers (MIPs) have focused mainly on the nature of heterogeneous binding sites. More data on the thermodynamics than on the kinetic features of MIP columns have been published. The present article addresses the sources of peak broadening and tailing, which are the main drawbacks often associated with imprinted polymers in chromatography for practical applications. With use of the theory of nonlinear chromatography, the peak properties of a MIP column, including the retention and peak broadening and tailing, can be well interpreted. Efforts to improve chromatographic efficiency using MIPs prepared by approaches different from the conventional method, including covalent imprinting and the format of uniformly sized spherical microbeads, are reviewed and discussed. This review leads to the conclusion that nonlinear chromatography theory is useful for characterizing chromatographic features of MIP columns, since a MIP is essentially an affinity-based chromatographic stationary phase. We expect more theoretical and experimental studies on the kinetic aspects of MIP columns, especially the factors influencing the apparent rate constant, as well as the analysis of the influences of mobile-phase composition on the chromatographic performance. In addition to revealing the affinity interaction by molecular recognition, slow nonspecific interactions which may be inherited from the imperfect imprinting and may be involved in the rebinding of the template to MIPs also need to be characterized. Figure The peak broadening and tailing associated often with molecularly imprinted polymers (MIPs) in column chromatography for practical applications can be well characterized by the theory of nonlinear chromatography.

Keywords: Molecularly imprinted polymers; Liquid chromatography; Nonlinear chromatography; Band broadening; Peak asymmetry

Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities by Toine F. H. Bovee; Willem G. E. J. Schoonen; Astrid R. M. Hamers; Marta Jorge Bento; Ad A. C. M. Peijnenburg (pp. 1111-1119).

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3′-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.

Keywords: Agonist; Antagonist; Plant hormones; Synthetic antiestrogens; Yeast estrogen bioassay; Yeast androgen bioassay

Development of electrochemical biosensors based on sol-gel enzyme encapsulation and protective polymer membranes by Rasa Pauliukaite; Monika Schoenleber; Pankaj Vadgama; Christopher M. A. Brett (pp. 1121-1131).

Protective polymer coatings have been used to enhance the retention of enzymes in sol-gel films as immobilisation phases in electrochemical biosensors. Carbon film electrodes were electrochemically modified with poly(neutral red) (PNR). These electrodes were coated with oxysilane sol-gels incorporating glucose oxidase and an outer coating of carboxylated PVC (CPVC) or polyurethane (PU), with and without Aliquat-336 or isopropyl myristate (IPM) plasticizer, was applied. The biosensors were characterised electrochemically using cyclic voltammetry and amperometry, electrochemical impedance spectroscopy and scanning electron microscopy. Impedance spectra showed that the electrode surface is most active when the sol-gel–GOx layer is not covered with a membrane. However, membranes without plasticizer extend the lifetime of the biosensor to more than 2 months when PU is used as an outer membrane. The linear range of the biosensors was found to be 0.05–0.50 mM of glucose and the biosensor with PU outer membrane exhibited higher sensitivity (ca.117 nA mM⁻¹) in the region of linear response than that with CPVC. The biosensors were applied to glucose measurement in natural samples of commercial orange juice.

Keywords: Carbon film electrode; Poly(neutral red); Biosensor; Amperometry; Cyclic voltammetry; Electrochemical impedance spectroscopy; Glucose determination; Polymer membrane

Rapid response behavior, at room temperature, of a nanofiber-structured TiO₂ sensor to selected simulant chemical-warfare agents by Xingfa Ma; Tao Zhu; Huizhong Xu; Guang Li; Junbao Zheng; Aiyun Liu; Jianqin Zhang; Huatai Du (pp. 1133-1137).

A chemical prototype sensor was constructed based on nanofiber-structured TiO₂ and highly sensitive quartz resonators. The gas-sensing behavior of this new sensor to selected simulant warfare agents was investigated at room temperature. Results showed rapid response and good reversibility of this sensor when used with high-purity nitrogen. This provides a simple approach to preparation of materials needed as chemical sensors for selected organic volatiles or warfare agents. Figure Sensing behavior of TiO₂ nanofiber sensor to chemical vapors

Keywords: Titanium dioxide; Nanofiber-structure; Chemical sensor; Chemical warfare agents; Organic volatiles

Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA by Ansgar Fitzner; Holm Frauendorf; Hartmut Laatsch; Ulf Diederichsen (pp. 1139-1147).

Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin–DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3′-end and an oligonucleotide with a phosphorylated 5′-end.

Keywords: DNA alkylation; DNA strand cleavage; Electrospray mass spectrometry; Gutingimycin; Trioxacarcin A

Bioaccessibility of selected trace metals in urban PM_2.5 and PM₁₀ samples: a model study by Thomas Falta; Andreas Limbeck; Gunda Koellensperger; Stephan Hann (pp. 1149-1157).

Bioaccessibility of trace metals originating from urban particulate matter was assessed in a worst case scenario to evaluate the uptake and thus the hazardous potential of these metals via gastric juice. Sampling was performed over a period of about two months at the Getreidemarkt in downtown Vienna. Concentrations of the assayed trace metals (Ti, Cr, Mn, Co, Ni, Cu, Zn, Mo, Ag, Cd, Sn, Sb, Tl and Pb) were determined in PM_2.5 and PM₁₀ samples by ICP-MS. The metal concentrations in sampled air were in the low picogram to high nanogram per cubic metre range. The concentrations in PM_2.5 samples were generally lower than those in PM₁₀ samples. The average daily intake of these metals by inhalation for a healthy adult was estimated to be in the range of <1 ng (Tl) to >1,000 ng (Zn). To estimate the accessibility of the inhaled and subsequently ingested metals (i.e. after lung clearance had taken place) in the size range from 2.5- to 10-μm aerodynamic equivalent diameter, a batch-extraction with synthetic gastric juice was performed. The data were used to calculate the bioaccessibility of the investigated trace metals. Extractable fractions ranged from 2.10% (Ti in PM_2.5) to 91.0% (Cd in PM_2.5), thus yielding bioaccessible fractions (PM_2.5–10) from 0.16 ng (Ag) to 178 ng (Cu).

Keywords: Bioaccessibility; Inductively coupled plasma mass spectrometry; Trace metals; Urban particulate matter; Synthetic gastric juice

Raman spectroscopic investigation of cocaine hydrochloride on human nail in a forensic context by Esam M. A. Ali; Howell G. M. Edwards; Michael D. Hargreaves; Ian J. Scowen (pp. 1159-1166).

This study describes the application of Raman spectroscopy to the detection of drugs of abuse and noncontrolled substances used in the adulteration of drugs of abuse on human nail. Contamination of the nail may result from handling or abusing these substances. Raman spectra of pure cocaine hydrochloride, a seized street sample of cocaine hydrochloride (77%), and paracetamol could be acquired from drug crystals on the surface of the nail. An added difficulty in the analytical procedure is afforded by the presence of a nail varnish coating the nail fragment. By using confocal Raman spectroscopy, spectra of the drugs under nail varnish could be acquired. Spectra of the drugs could be readily obtained nondestructively within three minutes with little or no sample preparation. Raman spectra could be acquired from drug particles with an average size of 5–20 μm. Acquisition of Raman point maps of crystals from both pure and street samples of cocaine hydrochloride under nail varnish is also reported. Figure Raman spectrum and point Raman map of cocaine HCI

Keywords: Cocaine hydrochloride; Nail; Forensics; Raman spectroscopy

A novel approach for monitoring of cyanobacterial toxins: development and evaluation of the passive sampler for microcystins by Jiří Kohoutek; Pavel Babica; Luděk Bláha; Blahoslav Maršálek (pp. 1167-1172).

We have investigated the ability of an integrative sampler for polar organic chemicals to sequestrate a group of common and highly hazardous cyanobacterial toxins—microcystins. In a pilot experiment, commercially available passive samplers were shown to effectively accumulate microcystins after 7 days’ exposure in the field. To find the most efficient configuration for sequestration of microcystins, four different porous membranes (polycarbonate, polyester, polyethersulfone and nylon) and two sorbents (Oasis HLB and Bondesil-LMS) were evaluated in the laboratory experiments, where samplers of different configuration were exposed to microcystins (microcystin-RR and microcystin-LR) for 14 days under steady conditions. We observed differences in sampling rates and amounts of accumulated microcystins depending on the sampler configurations. The samplers constructed with the polycarbonate membrane and Oasis HLB sorbent (2.75 mg/cm²) provided the highest sampling rates (0.022 L/day for microcystin-RR and 0.017 L/day for microcystin-LR). To the best of our knowledge, the present study is the first reporting application of passive samplers for microcystins, and our results demonstrate the suitability of this tool for monitoring cyanotoxins in water.

Keywords: Passive sampling; Cyanobacteria; Microcystin

In situ application of a cellulose bag and an ion exchanger for differentiation of labile and inert metal species in aquatic systems by Danielle Goveia; André Henrique Rosa; Iramaia Corrêa Bellin; Fabiana Aparecida Lobo; Leonardo Fernandes Fraceto; José Arnaldo Frutuoso Roveda; Luciane Pimenta Cruz Romão; Newton Luiz Dias Filho (pp. 1173-1180).

This work involved the development and application of a new analytical procedure for in-situ characterization of the lability of metal species in aquatic systems by using a system equipped with a diffusion membrane and cellulose organomodified with p-aminobenzoic acid groups (DM-Cell-PAB). To this end, the DM-Cell-PAB system was prepared by adding cellulose organomodified with p-aminobenzoic acid groups (Cell-PAB) to pre-purified cellulose bags. After the DM-Cell-PAB system was sealed, it was examined in the laboratory. The in-situ application involved immersing the DM-Cell-PAB system in two different rivers, enabling us to study the relative lability of metal species (Cu, Cd, Fe, Mn, and Ni) as a function of time and quantity of exchanger. The procedure is simple and opens up a new perspective for understanding environmental phenomena relating to the complexation, transport, stability, and lability of metal species in aquatic systems rich in organic matter.

Keywords: Lability; Metals; Aquatic organic matter; Water; Speciation

Whole-cell luminescence-based flow-through biodetector for toxicity testing by Philipp Stolper; Susanne Fabel; Michael G. Weller; Dietmar Knopp; Reinhard Niessner (pp. 1181-1187).

A new type of biodetector was designed based on a bioluminescence test with the bacterium Vibrio fischeri performed in a liquid continuous flow-through system. Here we describe the modification of a commercial tube luminescence detector to work in the flow mode by building a new flow cell holder and a new case including “top cover” to connect the flow cell with the waste and the incubation capillary in a light-proof manner. As different samples were injected successively it was necessary to keep the individual peaks separated. This was done using an air-segmented flow in the reaction coil. To afford fast screening, the incubation time of the sample and the Vibrio fischeri, which equaled the dead time of the detection system, was set at 5.6 min. Rapid monitoring of toxic substances is achieved by using 20 μL of sample and flow-rates of 110–150 μL min⁻¹. As a proof-of-principle, we show results for the detection of five selected di-, tri- and tetrachlorophenols at different concentrations varying from 1 to 200 mg L⁻¹. Calculation of inhibition rates and EC₅₀ values were performed and compared with corresponding values from the DIN EN ISO 11348-2 microplate format. Compared with the latter, the inhibition rates obtained with our flow-through biodetector for the compounds tested were generally about twofold lower, but importantly, a much faster detection is possible. Figure Flow scheme of the biodetector setup

Keywords: Biosensor; Air-segmented flow; Vibrio fischeri ; Bioluminescence; Toxicity test

Polyketide analysis using mass spectrometry, evaporative light scattering, and charged aerosol detector systems by Michael Pistorino; Blaine A. Pfeifer (pp. 1189-1193).

In this study, a mass spectrometer (MS), an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD) were used to analyze an erythromycin precursor (termed 6-deoxyerythronolide B). The work highlights the capabilities of each detector to analyze a representative polyketide compound that does not possess a natural chromophore, and presents the first comparison to include a charged aerosol system. Each detector was evaluated based upon limit of detection (LOD), dynamic range, and precision in the context of polyketide analysis. Due to its low LOD, wide dynamic range, and ability to provide molecular weight information, the MS was deemed the best detection option for the analysis of low-concentration, poorly identified polyketide compounds. Alternatively, both the CAD and ELSD systems studied showed better precision and accuracy. The ELSD demonstrated the best precision at 3%, but its LOD was limited to concentrations primarily greater than or equal to 1 mg/L. The Corona CAD demonstrated a LOD (0.012 mg/L) and dynamic range comparable to mass spectroscopy and therefore serves as a more cost-efficient alternative for polyketide production schemes with low titers.

Keywords: Polyketide; 6dEB; ELSD; Charged aerosol detector

Isolation and purification assay of ex vivo photosystem II D1 protein toward integrated biointeraction analysis by B. Muktiono; C. Schulten; O. Heemken; J. Gandrass; A. Prange; H. Schnabl; C. Cerboncini (pp. 1195-1202).

Protein extracts of photosystem II were prepared from leaf chloroplasts of different plant species by fast and nondenaturing methods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis of the proteins obtained showed that the extracts were enriched by D1 proteins, which appeared putatively in association with the 33-kDa oxygen-evolving-complex subunits. In further isolation steps D1 proteins were purified using salt-gradient chromatography (fast protein liquid chromatography) and characterized by western blot and mass spectrometry.

Keywords: D1 protein; Thylakoids; Oxygen-evolving complex

Rapid and quantitative evaluation of the effect of process variables on the kinetics of photocatalytic degradation of phenol using experimental design techniques and parallel factor (PARAFAC) analysis by Marta Bosco; M. Soledad Larrechi (pp. 1203-1207).

A 2³ factorial design has been used to analyze the effect of pH, the nature of the catalyst, and the concentration of the substrate on the rate constant of the photodegradation reaction of phenol. The main effects of the considered variables and their interaction are discussed. The significance of the effects has been corroborated using an ANOVA test. The values of phenol concentrations, used to calculate the rate constant, and the concentrations of intermediates were obtained by applying parallel factor (PARAFAC) analysis to the data obtained from monitoring the process by means of excitation–emission fluorescence (EEM). The proposed methodology, which combines experimental design and multivariate techniques, is a rapid alternative for study of chemical kinetics.

Keywords: Photocatalytic degradation; Parallel factor analysis; Experimental design; Emission–excitation matrix; Phenol

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Analytical and Bioanalytical Chemistry (v.390, #4)